Thrombocytopenia (low blood platelet levels) is most often caused either by defective platelet production or excessive platelet destruction. Defective platelet production is a common manifestation of many toxic, nutritional, and neoplastic disturbances of the bone marrow. Increased peripheral destruction of platelets is characterized by shortened platelet survival and increased proliferation of bone marrow megakaryocytes in an effort to compensate for the low platelet levels. Frequently, this process is immunologically mediated.
Certain drugs and their metabolites induce antibodies in some individuals which can cause immune platelet destruction. Implicated drugs include quinidine and quinine (stereoisomers of each other), sulfonamide antibiotics and many others (R. H. Aster, in Platelet Immunobiology: Molecular and Clinical Aspects. T. J. Kunicki and J. N. George eds., Lippincott, Philadelphia, pp. 387-435, 1989; N. R. Shulman, et al., "Platelet Immunology" in Hemostasis and Thrombosis: Basic Principles and Clinical Practice. R. W. Culman, J. Hirsh, V. J. Marder, E. W. Salzman, eds. Lippincott, Philadelphia, 2nd ed., pp. 452-529, 1989). A few of these drugs, such as penicillin, appear to bind covalently to platelet proteins and stimulate the formation of antibodies specific for the drug-protein complex (hapten-dependent antibodies) (D. J. Salamon, et al., Transfusion 24: 395, 1984). More often, however, the sensitizing drug or one of its metabolites induces the formation of antibody by an unknown mechanism (Aster, supra, 1989; A. Salama, et al., Sem. Hematol. 29: 54-63, 1992). The resulting antibodies bind to platelets only in the presence of drug to cause platelet destruction. Evidence obtained by the Applicants (D. J. Christie, et al., J. Clin. Invest. 75: 310, 1985; D. J. Christie, et al., J. Clin. Invest. 70: 989, 1982) and others (C. Mueller-Eckhardt, et al., Trans. Med. Rev. 4: 69, 1990; A. Salama, et al., Semin. Hematol. 29: 54, 1992) indicates that in such cases, the drug binds non-covalently and reversibly to selected platelet membrane proteins to induce conformational changes or form compound epitopes that are recognized by the antibodies. Drug-dependent binding of the antibodies to platelets causes the platelets to be destroyed. In the several forms of drug-induced immune thrombocytopenia, platelet counts are often very low and bleeding complications are frequently severe.
A third type of drug-induced thrombocytopenia (heparin-induced thrombocytopenia or HITP) occurs in patients treated with heparin to prevent or treat thrombosis. Heparin is a family of polysaccharide species consisting of chains made up of alternating, 1-4 linked and variously sulfated residues of glucuronic acid or iduronic acid and D-glucosamine. (B. Casu, "Methods of structural analysis" in Heparin: Chemical and Biological Properties, Clinical Applications, D. A. Lane and U. Lindahl, eds. CRC Press, Inc. Boca Raton, Fla., 1989, pp. 25-49.) In man and animal species, heparin is normally found in storage granules of mast cells (tissue basophils) (L. Enerback, "The mast cell system." In Heparin: Chemical and biological properties, clinical applications, D. A. Lane and U. Lindahl eds. CRC press, Inc., Boca Raton, Fla., pp. 97-114, 1989). Heparin-like molecules, such as heparan sulfate and chondroitin sulfate are expressed on the surface of endothelial cells that coat the luminal surface of blood vessels and in other tissues where they are coupled to a protein backbone (syndecan) to form a class of molecules known as proteoglycans (Ihrcke, et al., Immunology Today 14: 500-505, 1993). The heparin-like residues on endothelial cell proteoglycans are thought to provide one means by which abnormal clotting is prevented, allowing the circulating blood to remain in a fluid state (J. A. Marcum, et al., "The biochemistry, cell biology, and pathophysiology of anticoagulantly active heparin-like molecules of the vessel wall" in Heparin: Clinical and Biological Properties, Clinical Applications. D. A. Lane and U. Lindahl eds., CRC Press, Inc., Boca Raton, Fla., pp. 275-294, 1989). Heparin acts as an anticoagulant by binding to a co-factor protein, anti-thrombin III, in such a way as to enable this protein to inhibit certain activated clotting factors, especially activated Factor X (Xa) and thrombin (IIa) (I. Bjork, et al., "Molecular mechanisms of the accelerating effect of heparin on the reactions between antithrombin and clotting proteases" in Heparin: Chemical and Biological Properties, Clinical Applications, D. A. Lane and U. Lindahl eds., CRC Press, Inc., Boca Raton, Fla., pp. 229-255, 1989). Heparin of bovine origin appears to be more likely to cause HITP than heparin of porcine origin (W. R. Bell, et al., N. Engl. J. Med. 33: 902, 1980).
Thrombocytopenia in patients with HITP is usually not severe enough to result in bleeding. However, patients with this condition often experience thrombosis in major arteries and/or veins which can be fatal or cause the loss of a limb or a stroke. After discontinuation of heparin in patients with HITP, the platelet levels generally return to normal.
HITP appears to be caused by IgG or IgM antibodies that develop after five or more days of heparin therapy (G. P. Visentin, et al., J. Clin. Invest. 93: 81-88, 1994). These antibodies differ from those associated with other forms of drug-induced thrombocytopenia in that, in the presence of optimal concentrations of heparin, they activate blood platelets, causing the platelets to release the contents of their storage granules and to undergo membrane changes that create sites for the binding of a coagulation factor, fibrinogen, normally present in plasma (B. H. Chong, et al., Br. J. Haematol. 64: 347, 1986). The Applicants have shown that antibodies associated with HITP are specific for complexes of heparin and platelet factor 4 (PF4), a basic heparin-binding protein normally present in platelet storage granules (Visentin, et al., 1994, supra).
On the basis of findings made in their laboratory, the Applicants recently proposed the following new hypothesis to explain the development of thrombocytopenia and thrombosis in patients sensitive to heparin (Adapted from G. P. Visentin, et al. J. Clin. Invest. 93: 81-88, 1994): In a patient with IgG antibodies specific for heparin/PF4 complexes who is treated with heparin, a) minimal activation of circulating platelets by heparin alone (C. Eika, Scand. J. Hematol. 9: 480, 1972) or by immune complexes consisting of heparin, PF4, and IgG, leads to release of PF4 from platelet alpha-granules in a complex with chondroitin sulfate (S. Huang, et al., J. Biol. Chem. 257: 11546, 1982); b) circulating heparin displaces the chondroitin sulfate to form heparin/PF4 complexes (R. Handin, et al., J. Biol. Chem. 251: 4273, 198-); c) antibodies bind to heparin/PF4 to form immune complexes in close proximity to the platelet surface; d) these complexes bind to platelet Fc receptors, activate platelets, and release more PF4; e) the additional PF4 released reacts with heparin and IgG to form new immune complexes, promoting further platelet activation and causing thrombocytopenia; and f) PF4 released from platelets in excess of the amount that can be neutralized by available heparin binds to heparan sulfate on endothelial cells to create targets for IgG, IgA, or IgM antibodies leading to antibody-mediated endothelial injury and a predilection to thrombosis or disseminated intravascular coagulation. IgM antibodies, because of their greater capacity for complement activation, may be more destructive to endothelial cells than those of the IgG or IgA classes.
Because of the morbidity and mortality associated with HITP, it is important that the diagnosis be made quickly and accurately in a patient who develops thrombocytopenia while receiving heparin. Failure to make a diagnosis in such patients can lead to continuation of heparin therapy and fatal outcome. Assays used to diagnose other forms of drug-induced thrombocytopenia, i.e., binding of IgG or IgM antibodies to normal target platelets in the presence of drug (R. H. Aster, The Immunologic Thrombocytopenias in Platelet Immunology. T. J. Kunicki and J. N. George eds., Lippincott, Philadelphia, Pa., pp. 387-435, 1989) are not useful in detecting antibodies associated with HITP (G. P. Visentin, 1994, supra; H. C. Godal, "Heparin-induced thrombocytopenia" in Heparin: Chemical and Biological Properties, Clinical Applications, D. A. Lane and U. Lindahl eds., CRC Press, Inc., Boca Raton, Fla., pp. 533-548, 1989).
Accordingly, diagnostic techniques have been developed that make use of the ability of HITP-associated antibodies to activate platelets in the presence of optimum concentrations of heparin. One such test is the platelet aggregation test which is done by mixing the following reagents together in a test tube: normal platelet-rich plasma coagulated with citrate, heparin at a concentration of about one unit per ml, and plasma or serum from the patient suspected of having HITP. The mixture is incubated at 37.degree. C. and stirred. In a positive reaction, the antibody activates the platelets, causing the platelets to aggregate. The extent of aggregation is measured by an increase in light transmission through the mixture (J. G. Kelton, et al., Blood 72: 925-930, 1988). The assay is then repeated using a much higher concentration of heparin, e.g., 100 units per ml. Aggregation with the lower dose of heparin and lack of aggregation with the higher dose constitutes a positive test for HITP antibody.
A second and more sensitive test, also dependent on the ability of HITP antibodies to activate platelets, is the .sup.14 C-serotonin release test (D. Sheridan, et al., Blood 67: 27-30, 1986). In this assay, washed, normal donor platelets radiolabeled with .sup.14 C-serotonin are suspended in buffer and test serum. Heparin at a concentration of about 0.1 units per ml is then added and the mixture is agitated for about 30 minutes. In a positive test, .sup.14 C-serotonin is released from the platelets by virtue of their being activated by the HITP antibody (Sheridan, 1986, supra). As with the aggregation test, specificity of the reaction is confirmed by showing that .sup.14 C-serotonin release is inhibited by a high dose of heparin, e.g., 100 units per ml.
Another disclosed method is an assay for heparin-induced IgG antibodies based on their reaction with immobilized complexes of heparin and platelet factor 4 (PF4) (see Amiral, et al., Thromb. Haemostasis 68: 95-96, 1992). PF4 is a protein component of platelet alpha granules which is positively charged at neutral pH and is known to be capable of binding to and inhibiting the function of heparin. PF4 for use in the assay can be obtained by cleavage or lysis of normal platelets (see PCT Application W096/02833, 1992). PF4 belongs to a super-family of cytokines called "intercrines" or "chemokines" involved in the mediation of certain immune reactions and other activities (see Masushima, et al., Cytokines 1: 2-13, 1989). PF4 has high affinity for heparin (see Handin, et al., J. Biol. Chem. 251: 4273-4282, 1976) and is able to neutralize the anticoagulant properties of heparin (see Lane, et al., Biochem. J. 218: 725-732, 1984, Machalski, et al., Br. J. Haematol. 38: 561, 1978).
The heparin/PF4 assay described by Amiral (supra) is more convenient than the platelet aggregation test and the serotonin release test, which depend on activation of fresh platelets. However, discrepancies were observed when comparing results obtained with the heparin/PF4 assay with those obtained in a platelet aggregation test (see Greinacher, et al., Transfusion 34: 381-385, 1994). Thus, a method of diagnosing HITP that combines ease of use with high sensitivity and specificity as compared with the favored serotonin release test is not available.